腺病毒介导的人p27kip1基因转染对球囊损伤后兔颈动脉新生内膜增生的抑制作用

目的 研究腺病毒介导的p27kip1基因及其蛋白产物高表达对球囊损伤后兔颈动脉新生内膜增生的抑制作用。方法 建立兔颈动脉球囊损伤模型,将LacZ重组腺病毒（AdLacZ）和人p27kipl重组腺病毒（Adhp27kipl）在体内分别转染损伤动脉节段,以Western　blot、x-gal染色、HE染色、兔疫组化及计算机图像处理方法观察转染动脉节段中外源性p27kipl蛋白表达及其对新生内膜增生的影响。结果 与AdLacZ转染组及未转染组比较,Adhp27kip1转染的动脉节段内p27kip1蛋白呈高表达,表达高峰在7～14 d,持续4周以上;转染4周后未转染组、AdLacZ转染组及Adhp27kip1转染组的新生内膜面积分别为1.106mm2、0.988mm2及0.278mm2;管腔狭窄率分别为87.07％、65.40％及32.14％。结论 外源性p27kip1基因及其蛋白产物在损伤动脉节段内高表达可显著抑制新生内膜增生及管腔狭窄。     

Tacrolimus-eluting stent inhibits neointimal hyperplasia via calcineurin/NFAT signaling in porcine coronary artery model

AimsThe purpose is to elucidate the mechanism by which a newly developed tacrolimus-eluting stent (TES) prevents neointimal hyperplasia after stenting.Methods and resultsThe three major coronary arteries in juvenile swine were randomized to implantation of either a TES or bare metal stent (BMS). Twelve weeks after stenting, the TES showed 29% less neointimal area than the BMS. Immunohistochemical staining showed that the expression of calcineurin was up-regulated in the neointima and media after stenting, and the TES inhibited this up-regulation. Western blotting demonstrated that the expression of calcineurin, nuclear factor of activated T cell (NFAT), and interleukin-2 (IL-2) was lower with the TES than with the BMS. To confirm the effect of tacrolimus on vascular smooth muscle cells (VSMCs) and its mechanism, cultured rat VSMCs were incubated with 12.5 μM of tacrolimus (tacrolimus group) or without tacrolimus (control group). The cell number of the tacrolimus group was significantly lower than that of the control group at 48 h of incubation. Western blotting demonstrated that tacrolimus decreased the expression of calcineurin, NFATc4, and IL-2 of cultured VSMCs. We confirmed that calcineurin small-interfering RNA (siRNA) decreased cell proliferation and the expression of NFATc4 and IL-2 in cultured VSMCs compared with negative control-siRNA.ConclusionThe newly developed TES inhibited neointimal hyperplasia after stenting via the calcineurin/NFAT/IL-2 signaling pathway, which is one of several mechanisms through which TES inhibits restenosis. Calcineurin may be an important molecular target to prevent restenosis after stenting.     

Novel NAD(P)H oxidase inhibitor suppresses angioplasty-induced superoxide and neointimal hyperplasia of rat carotid artery

Neointimal proliferation occurring after vascular or endovascular procedures is a major complication leading to end-organ or limb ischemia. In experimental models, balloon injury has been shown to induce NAD(P)H oxidase to produce vascular superoxide anion (O2*-) production, which has been implicated in cell proliferation, but a direct link is still unclear. We postulated that inhibition of arterial NAD(P)H oxidase, resulting in decreased O2*-, would lessen the neointimal hyperplasia caused by balloon injury to the common carotid artery (CCA). Sprague-Dawley rats were implanted with osmotic minipumps containing either vehicle, a cell-permeant peptide that inhibits NAD(P)H oxidase (gp91ds-tat, 10 mg/kg per day), or a scrambled peptide control (scrmb-tat). Two days after pump implantation, the left CCA was injured using an intravascular balloon embolectomy catheter (2F Fogarty). Systolic blood pressure was monitored by tail cuff. Fourteen days after injury, CCAs were harvested and analyzed by digital morphometry. Rats in both groups remained normotensive, with no significant differences in systolic blood pressure. Reactive oxygen species measurements after injury indicated a significant reduction in vascular O2*- in rats infused with gp91ds-tat, and the neointima/media area and thickness ratios were significantly lower in their arteries compared with control. On the contrary, no significant change in overall CCA diameter was observed in any group. Our data indicate that in response to balloon injury of the rat carotid artery, NAD(P)H oxidase activity contributes to neointimal hyperplasia and is involved in vascular cell proliferation and migration during restenosis.     

过表达p27基因抑制血管损伤后新生内膜的形成

目的　探讨p2 7基因转染对血管损伤后新生内膜形成的影响。方法　以腺病毒为载体转染p2 7基因 ,用MTT法观察其对血管平滑肌细胞增殖的影响 ,用流式细胞仪检测细胞周期 ;在兔颈总动脉球囊损伤模型上转染p2 7基因 ,采用免疫组织化学法检测p2 7基因的表达 ,检测其对新生内膜形成的影响。结果　p2 7可明显抑制培养的血管平滑肌细胞增殖 ,细胞停滞于G1期。兔颈总动脉球囊损伤后转染p2 7基因可以减少新生内膜的形成达 2 7 4 3%。结论　以腺病毒为载体转染p2 7基因可以有效地抑制血管平滑肌细胞增殖及血管损伤后新生内膜的形成。     

Impact of gender on neointimal hyperplasia following coronary artery stenting

Whether gender affects long-term outcomes after bare metal stent implantation remains controversial. The aim of this study was to examine the impact of gender on neointimal hyperplasia in a large cohort of patients after stent implantation using 3-dimensional intravascular ultrasound. Lumen and stent areas were manually traced at 0.5-mm intervals throughout the stented segment. Using Simpson's method, lumen, stent, and neointimal (stent - lumen) volumes were calculated and standardized by stent length. Women were older, presented more often with hyperlipidemia or hypertension, and had smaller reference vessel diameter and mean stent area, compared with men. Although neointimal hyperplasia and neointimal thickness in women were similar to that in men, the percentage of neointimal hyperplasia (neointimal area divided by stent area) was higher in women due to the smaller stent area. After adjusting for stent area, the percentage of neointimal hyperplasia did not differ by gender. In conclusion, the results of this study indicate that neointimal hyperplasia after bare metal stent implantation in women is similar to that seen in men. Despite the similarity in outcome, there are several gender-specific differences in baseline characteristics.     

Macrophages,myofibroblasts and neointimal hyperplasia after coronary artery injury and repair

Macrophages participate in the restenosis process through the release of cytokines, metalloproteinases and growth factors. Studies of peritoneal granulation tissue suggest that macrophages may be precursors of myofibroblasts. This study examined the contribution of monocyte/macrophage lineage cells to neointimal cellular mass in a porcine model of thermal vascular injury. Thermal coronary artery injury caused medial smooth muscle cell necrosis and transformation of the media into an extracellular matrix barrier. The neointimal hyperplasia that developed over the injury sites was evaluated by light microscopy, electron microscopy and immunohistochemistry. At day 3, blood monocytes were adhered to the vessel wall and infiltrated the fibrotic media. At day 14, 42+/-3.9% of neointimal cells had a monocytic nuclear morphology and expressed macrophage-specific antigen SWC3 (identified by monoclonal antibody DH59B). Moreover, 9.2+/-1.8% of neointimal cells co-expressed SWC3 and alpha-smooth muscle actin and had ultrastructural characteristics intermediate between macrophages and myofibroblasts. At day 28, 10.5+/-3.5% of cells expressed SWC3 and 5.2+/-1.8% of cells co-expressed SWC3 and alpha-smooth muscle actin. This study indicates that hematopoietic cells of monocyte/macrophage lineage abundantly populate the neointima in the process of lesion formation and may be precursors of neointimal myofibroblasts after thermal vascular injury.     

Elevated potassium intake inhibits neointimal proliferation in the swine coronary artery

Background:Previously, we reported that elevated extracellular potassium concentration in vitro inhibited proliferation and migration of vascular smooth muscle cells, formation of free radical compounds by macrophages, and reduced platelet sensitivity to agonists. More recently, we described a reduction in neointimal proliferation after balloon angioplasty injury in the carotid arteries of rats associated with an elevation of dietary potassium intake during a 4-week experiment. In the present study we conducted a similar investigation in the swine coronary artery balloon angioplasty model.Procedures:Two groups of seven castrated male swine were studied; for 28 days the normal potassium group consumed a diet containing 0.25% potassium and the high potassium group ate diet containing 2.0% potassium. After 14 days on the diet, balloon angioplasty was performed. After an additional 14 days on the same diets the hearts were removed, and normal and lesioned sections of the artery were analyzed histologically.Results:The neointimal area was markedly less in the high potassium group than in the normal potassium group, 0.33 ± 0.04 mm² v 0.74 ± 0.10 mm² (P < .004). Neointimal area-to-total wall area ratio in the normal potassium group averaged 0.199 ± 0.018, significantly greater than the ratio computed for the elevated potassium group, 0.120 ± 0.015 (P < .006).Conclusion:These results support the hypothesis that a high level of dietary potassium intake inhibits neointimal proliferation after balloon angioplasty in the swine coronary artery.     

Zotarolimus-eluting stents reduce experimental coronary artery neointimal hyperplasia after 4 weeks.

AIMS: The addition of drug elution to coronary stents plays an integral role in coronary restenosis prevention. The present study was undertaken to determine the mechanism of action and the in vitro and in vivo efficacy of zotarolimus, a new chemical entity designed specifically for elution from phosphorylcholine (PC)-coated stents, for the reduction of neointimal hyperplasia in porcine coronary arteries. METHODS AND RESULTS: In vitro studies of Zotarolimus bound to FKBP-12 potently inhibited smooth muscle cells (SMCs) and endothelial cell (EC) proliferation. Twenty PC-only and 20 stents eluting zotarolimus 10 microg/mm were implanted in the coronary arteries of 20 domestic juvenile swine. After 28 days, zotarolimus stents exhibited less area stenosis (22.4+/-8.6 vs. 35.7+/-13%, P = 0.01), less neointimal area (1.69+/-0.55 vs. 2.78+/-1.07 mm(2), P = 0.01), less neointimal thickness (0.25+/-0.07 vs. 0.38+/-0.13 mm, P = 0.01), and greater lumen area (6.07+/-1.39 vs. 5.02+/-1.3 mm2, P = 0.01). All arteries in both the polymer-only and polymer/drug stent showed near-complete healing and minimal toxicity. Zotarolimus did not affect the extrastent segments nor alter the overall artery size (external elastic lamina cross-sectional area 9.18+/-1.19 vs. 9.06+/-1.28 mm2, P = 0.7). CONCLUSION: Zotarolimus binds to FKBP-12 and in vitro inhibits SMC and EC proliferation. Zotarolimus applied to PC-coated stents reduces neointima in the swine coronary model after 28 days. These results suggest potentially promising human clinical application for coronary stenting with this polymer/drug combination.     

Alpha-lipoic acid inhibits fractalkine expression and prevents neointimal hyperplasia after balloon injury in rat carotid artery

Vascular inflammation induced by the proinflammatory cytokine/NF-kappaB pathway is one of the key mechanisms in the development of neointimal hyperplasia. Accumulating evidence suggests that a recently identified chemokine, fractalkine, is involved in arterial inflammation and atherogenesis. However, no study has examined the expression of neointimal fractalkine and the effects of pharmacological agents on this process. The purposes of this study were to measure neointimal fractalkine expression in the rat carotid artery following balloon injury and to determine if alpha-lipoic acid (ALA) inhibits fractalkine expression and neointimal hyperplasia. Balloon injury of the rat carotid artery induced fractalkine expression in the medial as well as neointimal regions. ALA inhibited this expression and consequently prevented neoinitmal hyperplasia in a balloon-injured rat carotid artery. Additionally, ALA inhibited TNF-alpha-stimulated fractalkine expression in cultured vascular smooth muscle cells (VSMCs), a process which is mediated through the NF-kappaB pathway. In addition to fractalkine, ALA successfully inhibited TNF-alpha-stimulated expression of vascular cell adhesion molecule-1 and monocyte chemotactic protein-1 in cultured VSMCs. These data suggest that the cytokine-fractalkine system is involved in the pathogenesis of restenosis. The present study supports the possibility that ALA, which inhibits the NF-kappaB/fractalkine pathway, may be used to prevent neointimal hyperplasia after angioplasty or stenting.     

Effect of external beam irradiation on neointimal hyperplasia after experimental coronary artery injury.

Human coronary artery restenosis after percutaneous revascularization is a response to mechanical injury. Smooth muscle cell proliferation is a major component of restenosis, resulting in obstructive neointimal hyperplasia. Because ionizing radiation inhibits cellular proliferation, this study tested in a porcine coronary injury model the hypothesis that the hyperplastic response to coronary artery injury would be attenuated by X-irradiation. Deep arterial injury was produced in 37 porcine left anterior descending coronary artery segments with overexpanded, percutaneously delivered tantalum wire coils. Three groups of pigs were irradiated with 300-kV X-rays after coil injury: Group I (n = 10), 400 cGy at 1 day; Group II (n = 10), 400 cGy at 1 day and 400 cGy at 4 days and Group III (n = 9), 800 cGy at 1 day. Eight pigs in the control group underwent identical injury but received no radiation. Treatment efficacy was histologically assessed by measuring neointimal thickness and percent area stenosis. Mean neointimal thickness in all irradiated groups was significantly higher than in the control groups and thickness was proportional to X-ray dose. X-irradiation delivered at these doses and times did not inhibit proliferative neointima. Rather, it accentuated the neointimal response to acute arterial injury and may have potentiated that injury.     

Probucol inhibits in-stent thrombosis and neointimal hyperplasia by promoting re-endothelialization

BACKGROUND: Evidence suggests that delayed re-endothelialization is responsible for in-stent thrombosis. Probucol inhibits neointimal thickening in animals via enhanced re-endothelialization and is the only oral drug that consistently inhibits restenosis after coronary angioplasty in humans. Here, we examined the effects of probucol on re-endothelialization and neointimal formation in a stent model. METHODS AND RESULTS: New Zealand White rabbits were fed a hypercholesterolemic diet with probucol (1%) or without (control) (n=11 each) for 6 weeks. At 2 weeks, endothelial denudation and stenting of the iliac artery was performed. Iliac arteries were harvested at week 6, and stented segments sectioned and analyzed. Compared with control, probucol increased in-stent re-endothelialization (74+/-6% in controls versus 93+/-3% in probucol-treated; P=0.008), and decreased average luminal stenosis (58+/-27 versus 31+/-16%; P=0.01) and stent depth (619+/-310 versus 314+/-158 microm; P=0.009). Compared with control, probucol also decreased accumulation of macrophages in the neointima. Furthermore, none of the probucol-treated rabbits had in-stent thrombosis, whereas four of eleven control rabbits showed thrombosis (P=0.04). CONCLUSIONS: Probucol demonstrates anti-restenotic and appears to have anti-thrombotic properties that are likely related to its ability to promote in-stent re-endothelialization.     

High-dose granulocyte-colony stimulating factor promotes neointimal hyperplasia in the early phase and inhibits neointimal hyperplasia in the late phase after vascular injury

BACKGROUND: Granulocyte-colony stimulating factor (G-CSF) affects injured arteries through early endothelialization. Some reports, however, have cautioned that the restenosis rate may increase after G-CSF injection. In the present study, high-dose G-CSF was administered to mice with vascular injury to clarify its effect. METHODS AND RESULTS: Mice were received daily subcutaneous injections of saline or a high dose (300 microg/kg) of G-CSF for 5 days after vascular injury. In the FACS analysis, CD34-/Sca-1-positive progenitor cells were more abundant in the G-CSF group (p<0.05). Neointimal hyperplasia was more evident in the G-CSF group at 1 week (p<0.05), whereas at 4 weeks it was more evident in the control group (p<0.01). TUNEL-positive cells in the arterial wall were more numerous in the G-CSF group at day 1 (p<0.01). CD34-positive cells were observed in the G-CSF group at 1 week. Re-endothelialization appeared earlier in the G-CSF group (at 4 weeks; p<0.01). An increased number of 1A4-positive smooth muscle cells were found in bone marrow cell culture treated with G-CSF. CONCLUSION: High-dose G-CSF induced neointimal proliferation through excessive inflammation and bone marrow cell mobilization in the early phase. In the late phase, however, it induced early re-endothelialization and thereby inhibited neointimal hyperplasia.     

Effect of cilostazol on in-stent neointimal hyperplasia after coronary artery stenting: a quantative coronary angiography and volumetric intravascular ultrasound study.

BACKGROUND: This study was designed to investigate the efficacy of cilostazol on the prevention of in-stent neointimal hyperplasia as measured by both quantitative coronary angiography (CAG) and volumetric intravascular ultrasound (IVUS). METHODS AND RESULTS: Fifty-nine patients (39 men, age 62 years) undergoing elective coronary stenting were randomly assigned to receive aspirin plus clopidogrel or ticlopidine (Group I, n=28, 30 lesions) or aspirin plus clopidogrel or ticlopidine plus cilostazol (Group II, n=31, 35 lesions). CAG and IVUS were performed and repeated at 6 months to assess the primary endpoints of minimal luminal diameter (MLD) and in-stent neointimal hyperplasia volume. Follow-up CAG was performed on all patients and follow-up IVUS study was available for 50 lesions in 48 patients (24 lesions in Group I, 26 in Group II). There were no significant differences in the baseline angiographic data between the 2 groups. At 6 months follow-up, in-stent MLD was 1.90+/-0.76 mm in Group I and 2.41+/-0.85 mm in Group II (p=0.006). Volumetric IVUS at 6 months demonstrated that in-stent intimal hyperplasia volume per stent length was 2.2+/-1.4 mm3/mm in Group I and 1.0+/-0.5 mm3/mm in Group II (p=0.001). CONCLUSIONS: Triple antiplatelet therapy including cilostazol seems to be more effective at preventing in-stent neointimal hyperplasia than a dual antiplatelet regimen.     

Absence of p16 and p27 gene rearrangements and mutations in de novo myelodysplastic syndromes

Myelodysplastic syndromes (MDS) represent a group of clonal hematopoietic disorders characterized by dyshemopoiesis and frequent evolution to acute leukemia. Tumor suppressor gene inactivation may be involved in MDS pathogenesis. The two families of cyclin-dependent kinase inhibitors (CDKIs) (INK4 family of p15, p16, p18 and p19 and CIP/KIP family of p21, p27 and p57) that negatively regulate cell cycle progression are known tumor suppressor genes. To determine whether genetic alterations of p16 and p27 genes play an important role in MDS pathogenesis, we examined DNA from 51 patients classified as 17 refractory anemias (RA), four refractory anemias with ringed sideroblasts (RARS), 19 refractory anemias with an excess of blasts (RAEB), 5 refractory anemias with excess of blasts in transformation (RAEB-t) and 6 chronic myelomonocytic leukemias (CMML). Southern blot analysis detected no homozygous deletions of p16 and p27. Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and sequencing did not reveal point mutations for both genes with the exception of two allelic polymorphisms, namely a C --> G transition at 447 bp of p16exon3 and a T --> A transition at 791 bp of p27exon1 genes. Our results suggest that mutations of p16 and p27 genes resulting in abnormal p16 and p27 proteins do not represent a mechanism of gene inactivation involved in the pathogenesis of MDS.